Oil-in water emulsions

ABSTRACT

PCT No. PCT/SE95/00115 Sec. 371 Date Jul. 15, 1996 Sec. 102(e) Date Jul. 15, 1996 PCT Filed Feb. 6, 1995 PCT Pub. No. WO95/20943 PCT Pub. Date Aug. 10, 1995The invention relates to an oil-in-water emulsion comprising 0.01-50% by weight of the total preparation, preferably 0.1-10%, of a galactolipid material as an emulsifier. The galactolipid material consists of at least 50% digalactosyldiacylglycerols, the remainder being other polar lipids. The said emulsion is suitable as a carrier for one or more active substances in a pharmaceutical composition, but also in nutritional, cosmetical, food and agricultural products.

TECHNICAL FIELD

This invention relates to oil-in-water type emulsions comprising a polarlipid material as an emulsifier. These emulsions are suitable for use ascarriers for an active substance in a pharmaceutical composition, butalso in nutritional, cosmetical, food and agricultural products.

1. Background of the Invention

Emulsions of the type oil-in-water for pharmaceutical applications, suchas clinical nutrition and for the administration of lipophilic drugs,are generally based on natural lipids. The oil is typically a vegetableoil such as soybean oil, safflower oil or medium-chain triacylglycerol(MCT) oil. The emulsifier is typically a phospholipid such as egg yolkphospholipids (egg lecithin) or soybean phospholipids (soy lecithin).These emulsifiers consist of mixtures of phospholipid classes, such asphosphatidylcholine and phosphatidylethanolamine, which arezwitter-ionic, and phosphatidylinositol, which is anionic. It is widelycommon knowledge that these lecithin emulsifiers are the most utilisednatural lipids in preparing emulsions on an industrial scale of the kindmentioned above. It is also well-known that such emulsions suffer fromdisadvantages and problems which relate to the emulsifier beingphospholipids. Such disadvantages and problems are, for example, broadparticle size distributions and particle fusions resulting in so-calledcreaming.

Most commercial fat emulsions are based on egg phospholipids, which areproduced from animal sources, in most cases egg yolk powder. Animalsources are, in some cases, related to problems connected to viruscontamination, and, in the specific case of egg yolk powder, bacteriasuch as Salmonella. Another important feature of egg phospholipids isthe content of polyunsaturated fatty esters, such as arachidonate anddocosahexaenoate, which are extremely susceptible to oxidation in thepresence of even small amounts of oxygen. Thus, the odour and taste ofegg phospholipids are often very unpleasant, which can be carriedthrough to the fat emulsions. Contamination and oxidation may oftencause problems which relate to industrial safety and handling aspects.

2. Prior Art

EP-A2-0 402 090 discloses an edible oil-in-water emulsion suitable forcreams and dressings comprising 10-99% of the entire oil and fat contentof a diglyceride mixture. In order to improve the stability the emulsioncan also include 0.1-10%, based on the oil phase, of phospholipids.

EP-A2-0 391 369 discloses a stable pharmaceutical composition of anoil-in-water emulsion type which comprises an effective amount of alipophilic drug. The emulsion is composed of 3-50% of an oily carrier,mainly MCT oil, 0.05-20% of a phospholipid, 0.03-10% of a non-ionicsurfactant, and 0.05-50% of an ionic surfactant. The improved stabilityis said to be caused by a synergism between the stated ingredients.

Glycosylglycerides are a type of glycolipids which are well-knownconstituents of plant cell membranes. Two types based on galactose arevery common, monogalactosyldiacylglycerol, MGDG, anddigalactosyldiacylglycerol, DGDG, representing up to 40% of the dryweight of the thylakoid membranes.

Plant glyoolipids have carbohydrate units, mainly of galactose, linkedto glycerol. In MGDG the 1-position of the galacrose ring has a β-linkto glycerol, and in DGDG there is an α,1-6 bond between the sugars. Aminor constituent is the plant sulpholipid, more correctly namedsulphoquinovosyldiacylglycerol, SQDG, which contains a sulphonate ratherthan a hydroxyl group linked to carbon 6 of the terminal deoxyglucoseresidue. Most plant glycolipids can be described by the general formula##STR1## wherein R₁ and R₂ independently of each other are saturated orunsaturated fatty acid residues of 2-24 carbon atoms and 0-6 doublebonds, further esterified hydroxy acids, that is estolides, or hydrogen;the carbohydrate is a monosaccharide unit; n=1-5; and R₃ is a hydroxylor sulphonate group.

In investigating the interaction of glycosylglycerides with water andother polar solvents we have surprisingly found that specific glycolipidmaterials from cereals have a behaviour which makes said lipid materialssuitable and simple to utilise as a carrier material especially forpharmaceutical compositions, and also for other formulations, such ascosmetical, agricultural, nutritional and food applications.

SE 9400368-8 discloses an industrially applicable process for preparinga glycolipid material from plants, preferably cereals, by means ofextraction and chromatographic separations. The glycolipid material soprepared can be used as an amphiphilic material in pharmaceuticalproducts, cosmetics and food.

DESCRIPTION OF THE INVENTION

This invention relates to an oil-in-water emulsion comprising 0.01-50%by weight of the total preparation, preferably 0.1-10%, of an emulsifierand 0.1-70% by weight of the total preparation of an oily materialemulsified in an polar solvent, characterised in that the emulsifier isa galactolipid material consisting of at least 50%digalactosyldiacylglycerols, the remainder being other polar lipids.

In a preferred preparation the galactolipid material consists of about70-80% digalactosyldiacylglycerols and 20-30% other polar lipids.

In another preferred preparation the galactolipid material consists ofup to 100% digalactosyldiacylglycerols.

The digalactosyldiacylglycerols can be described by the general formula##STR2## wherein R₁ and R₂ independently of each other are saturated orunsaturated fatty acid residues of 10-22 carbon atoms and 0-4 doublebonds, or hydrogen; and R₃ is a hydroxyl or sulphonate group.

As preferred examples of fatty acid residues R₁ and R₂ can be mentionednaturally occurring fatty acyl groups, such as residues from thesaturated acids palmitic (C₁₅ H₃₁ CO; 16:0) and stearic acid (C₁₇ H₃₅CO; 18:0); from the monounsaturated acid oleic acid (C₁₇ H₃₃ CO; 18:1);and from the polyunsaturated acids linoleic (C₁₇ H₃₁ CO; 18:2) andlinolenic acid (C₁₇ H₂₉ CO; 1813). The fatty acid residues can alsocontain hydroxy acids linked to the glycerol moiety with their hydroxylgroups esterified by further fatty acids, so called estolides.

The other polar lipids being part of the galactolipid material are amixture of different glyco- and phospholipids, such as MGDG andphosphatidylcholines. The composition depends on the starting materialand process used for the manufacture of the galactolipids.

The specific proportions of the components of the galactolipid materialare not critical to the present invention as long as the content of DGDGis at least 50%. For many applications, however, the maximum benefitsare realised by a high content of DGDG, the most importantbilayer-forming component.

The galactolipid material can be extracted from almost any kind of plantmaterial. Preferred plant materials are seeds and kernels from grainsand cereals, for instance wheat, rye, oats, corn, rice, millet andsesame. Oat groats as well as wheat gluten have a high lipidconcentration and are therefore of an advantage to use in the process ofpreparation. The digalactosyldiacylglycerols of the galactolipidmaterial can, if applicable, also be of synthetic origin.

The oily material is any lipophilic material having a liquid orsemi-solid consistency at room temperature. No particular limitation isimposed on the oily material. Plant oils, animal oils, synthetic oils,fatty acids, natural synthetic glycerides, and lipophilic drugs, etc.,may be mentioned by way of example.

Preferred oils are plant oils containing γ-linolenic acid (GLA), such asevening primrose oils and borago oil, and fish oils containingeicosapentaenoic acid (EPA) and docosahexanoic acid (DHA).

The ratio between emulsifier and oily material could preferably bewithin the range of 1:40-1:10 by weight, especially 1:25-1:15 by weight.

An intrinsic beneficial feature of the galactolipids is the galactoseunits comprising the polar headgroup in each lipid molecule, which maysterically stabilise the emulsion droplets, and thus provide for aprolonged life-span when injected into the bloodstream.

Synthetic diglycosyldiacylglycerols based on galactose or any othermonosaccharide unit, such as glucose, and natural glycosylglycerides,isolated from any source, based on other carbohydrate units thangalactose, such as glucose, can be used in accordance with theinvention.

The oil-in-water emulsions of the invention are prepared by using thegalactolipid material as the emulsifier but may contain otherlow-molecular compounds in an effective isotonic amount. Theoil-in-water emulsion may also comprise optional additives known in theart for improving different aspects of the composition, such asflavouring agents, colorants, thickening agents, co-surfactants,preservatives, antioxidants, etc.

The emulsions are prepared by conventional methods. For example, a 30%(w/w) emulsion of medium-chain triacylglycerol oil in water is preparedby dispersing the emulsifier, that is the galactolipid material, in theoil. Glycerol and water are mixed. The oil phase as well as the aqueousphase are preheated and then the oil phase is added to the aqueous phaseunder high shear mixing. It is then subjected to high-pressurehomogenisation.

The invention also refers to a pharmaceutical composition comprising atherapeutically active substance in combination with the oil-in-wateremulsion.

The therapeutically active substance can be a lipophilic drug such asanti-cancer agents, anti-microbial and particularly anti-fungal agents,immunosuppressant drugs like cyclosporin, dermatological drugs,psychotropic drugs, anaestethic drugs and other drugs which arelipophilic and which may present formulation problems which could besolved by the use of galactolipids.

A preferred oily material for the emulsion is in addition to theprevious mentioned preferred oils also an MCT oil. There are also manylipids such as free fatty acids, mono-, di- and triacylglycerols,phospholipids, cholesterol esters and lipids of many other types whichhave therapeutic actions in themselves and which may be advantageouslyformulated in the form of an emulsion, based on the galactolipids. Inthis case the therapeutically active substance is the oily material,which can also have other bioactive properties.

A pharmaceutical composition can be as follows:

a therapeutically active substance in a therapeutically effectiveamount;

a galactolipid emulsifier, 0.1-5.0% by weight of the total composition;

an oily material, 1-50% by weight of the total composition;

optionally, an isotonic agent in an isotonically effective amount.

The isotonic agent is, for example, glycerol but could also be anyisotonic agent in an isotonically effective amount.

The polar solvent can be water or aqueous solutions, such as buffers andsaline, or any other conventional solvent, such as ethanol, glycerol,propylene glycol, polyethylene glycol, polypropylene glycol, glycofuran,methyl pyrrolidone, transcutol. Water is however the preferred solvent.

A pharmaceutical composition for parenteral administration can be asfollows:

0.2-3% 2,6-diisopropylphenol,

0.3-5% galactolipid material,

5-30% triacylglycerol oil,

an isotonically effective amount of an isotonic agent,

ad 100% water.

The pharmaceutical composition may be formulated for oral, enteral,parenteral, rectal, vaginal, topical, ocular, nasal or auraladministration to animals, especially mammals, including humans.

The emulsions based on the galactolipids are surprisingly stablepreparations compared to phospholipid emulsions made from egg lecithinor soy lecithin. Shaking tests, which destroy phospholipid emulsions,have no effect on galactolipid emulsions.

The galactolipid emulsions also exhibit a narrow and consistent particlesize distribution, which normally is a problem with the phospholipidemulsions. Commercially available fat emulsions based on egg lecithinoften have a problem of containing particles that are too large, whichmay result in problems such as the creaming phenomenon or the appearanceof oil droplets on the surface.

The galactolipid emulsions are also surprisingly stable to sterilisationby autoclaving in a standard autoclave. Commercially available fatemulsions often need to be autoclaved in special rotating autoclaves,which poses a technical problem. The use of standard autoclavingprocedures is a pronounced industrial improvement provided by theinvention.

Galactolipid Material

Galactolipid materials have been prepared from different cereals asstated below, and used for making carrier preparations andpharmaceutical compositions of the invention as stated in the examples.In the specification % refers to % by weight if not otherwise stated.The proportion of the solvents in solvent mixtures is given in parts byvolume.

Galactolipid Material from Oats

200 kg of oat kernels (Kungsornen AB, Sweden) were ground and extractedwith 1000 l of 95% ethanol at 70° C. for 3 h in an extraction tank understirring. The slurry was centrifuged while still warm and separated fromsolid particles. The liquid fraction was evaporated at 60° C. which gaveabout 10 kg of a light brown oil.

The oil was applied to a stainless steel column containing 6.25 kg ofsilica gel (Martex Silica Si, particle size 20-45 pore diameter 60 Å,from Amicon Corp., USA). The column temperature was 50° C. The columnwas then washed with 30 l of a mixture of hexane:isopropanol, 90:10, inorder to remove all nonpolar lipids.

The galactolipid material was then eluted from the column with 20 l of amixture hexane:isopropanol, 60:40, giving a galactosyldiacylglycerolfraction. Evaporation of this fraction gave about 700 g of DGDG, themajor lipid class. The galactolipid material was then dispersed in waterand subjected to freeze-drying, which resulted in a free-flowing powder.

Enrichment of DGDG from Galactolipids

50 g galactolipids from oats, as obtained above, having a content ofDGDG of about 70%, were dissolved in 250 ml hexane: isopropanol, 70:30,giving a total amount of 300 ml. The solution obtained was loaded on asilica gel (110 g) column and the less polar constituents were elutedwith 1 l of the mixture of hexane:isopropanol, 70:30. The enriched DGDGfraction was eluted with 2 l acetone. The acetone was evaporated andfreeze-dried. The total yield was 17 g of an almost pure DGDG product.

Hydrogenation of Galactolipids

200 g of a galactolipid mixture obtained from oats as stated above wasdissolved in 2 l warm isopropanol. 15 g of a palladium on carboncatalyst (Pd 15%, moisture 53%, Engelhard Rome s.r.i., Italy) was placedin the bottom of a pressure reactor (Model No. 4552M; Parr InstrumentCo., USA) equipped with two impellers on a stirrer shaft. The solutionwas then transferred into the reactor under a seal of nitrogen to reducethe fire hazard. The reactor vessel was sealed and first pressurizedthree times with nitrogen in order to remove air and then three timeswith hydrogen gas (Plus 4.5, from AGA Gas AB, Sweden). The hydrogenpressure was then kept at 6 bars, the stirrer set at 600 rpm and themixture was heated to 70° C. It took 14 minutes for the reaction mixtureto reach its temperature setpoint. The hydrogenation process wasconducted for 6 hours after which the reaction product was filteredthrough a 0.45 μm filter in order to remove carbon particles andpalladium. Solvent was evaporated on a rotavapor, the residual solidmaterial was dispersed in 1600 ml of deionized water and freeze-dried.

The yield of hydrogenated galactolipids after filtration andfreeze-drying was 155 g. The hydrogenation performance was evaluated bygas chromatography; only saturated fatty acids could be detected in thehydrogenated product.

Galactolipids from Wheat Gluten

1 kg of wheat gluten powder (AB Skanebrannerier, Sweden) was extractedwith 4 l of 95% ethanol at 70° C. for 3 h in a beaker. The slurry wasthen faltered under a pressure of 400-500 kPa and the filtercakeobtained was washed with 1 l of warm 95% ethanol. The combined ethanolsolutions were evaporated at maximum 60° C. and gave about 60 g of ayellow oil.

The oil was applied to a stainless steel column containing 45 g ofsilica gel (Martex Silica Si, particle size 20-45 μm, pore size 60 Å,from Amicon Corp., USA). The column was then washed with 700 ml of amixture hexane:isopropanol, 90:10, in order to remove neutral lipids.

In order to remove MGDG and some other polar lipids the column wassubsequently washed with 1000 ml of a mixture hexane: isopropanol,70:30. Elution of DGDG was carried out with 1000 ml of pure acetone.After evaporation about 4 g of an almost pure DGDG product was obtained.

Galactolipids from Rye

100 g rye flakes (Kungsornen AB, Sweden) were stirred for 60 min in amixture of industral hexane and isopropanol, 90:10. The slurry wasfiltered and evaporated which gave 0.5 g polar lipids. The residue,dissolved in 10 ml of a mixture of hexane and iso-propanol, 70:30, wasloaded on three Sep-pak Silica plus columns (Millipore Corp., USA)connected in series, washed with 20 ml of the same mixture of solventsand eluted with 15 ml acetone. The eluate was evaporated andfreeze-dried and the yield was 47 mg of galactolipids.

Chemical and Physical Characterization of Different GalactolipidMaterials Lipid Class Analysis

Lipid class analysis was performed by high performance liquidchromatography, HPLC, using a column packed with diol-modified silica(LiChrosphere 100 DIOL, 5 μm, 250 mm×4 mm i.d.; E. Merck, Germany). Thecolumn was enclosed in a water bath held at 75° C. The analytical systemconsisted of a HPLC pump CM 4000 (LDC/Milton Roy, USA), and an injector,model 7125, with a 20 l injection loop (Rheodyne Inc., USA). Theevaporative light-scattering detector used was a Sedex 45 (S.E.D.E.R.E.,France) equipped with a Sedex 55 nebulisation chamber with a drift tubetemperature and air inlet pressure of 97° C. and 2.0 bar, respectively.

The flow of the mobile phase was 1 ml/min during the analysis. A binarysolvent gradient, linear over 25 min, was used starting with 100% of Aand ending with 100% of B, whereA=hexane:isopropanol:n-butanol:tetrahydrofuran:isooctane:water,64:20:6:4.5:4.5:1, andB=isopropanol:n-butanol:tetrahydrofuran:isooctane:water,75:6:4.5:4.5:10. All solvents contained ammonium acetate, 180 mg/l.

Data collection and processing were done with GynkoSoft Data systemversion 4.22 (Softron GmbH, Germany). Typical amount injected foranalysis was 100 μg. Identification was based on retention timecomparison with authentic standards (Karlshamns LipidTeknik AB, Sweden).Volatile compounds were not detected in this system. Quantification wasbased on peak area calculations.

Zeta potentials were determined on dilute aqueous galactolipiddispersions with a Zetasizer 4 instrument (Malvern Instruments Ltd., UK)

                  TABLE 1                                                         ______________________________________                                        Characterisation of different galactolipid materials                          o-GL         o-h-GL  o-DGDG   w-GL w-DGDG r-GL                                ______________________________________                                        DGDG   73     70     72    100    80   100    67                              content,                                                                      area %                                                                        Z-poten-                                                                             -74    -76    -30   -51    -75  -38    -37                             tial, mV                                                                      ______________________________________                                    

In this Table 1 as well as in Table 2 below the following abbreviationsare used

o-GL=galactolipids from oats

o-h-GL=hydrogenated galactolipids from oats

o-DGDG=enriched galactolipids from oats

w-GL=galactolipids from wheat

w-DGDG=enriched galactolipids from wheat

r-GL=galactolipids from rye

Fatty Acid Analysis

Analysis of the fatty acid profile was done by gas chromatography aftertransesterification of the lipids to fatty acid methyl esters. Thesewere separated and quantified by capillary column gas chromatography ona Varian 3500 Capillary Gas Chromatograph equipped with a capillarycolumn 30 m×0.25 mm i.d. (DB-WAX; J&W Scientific, USA), an on-columninjector and a flame ionization detector. Helium was used as the carriergas. Integration was performed with GynkoSoft Data system version 4.22(Softton GmbH, Germany). Transesterification was done by adding 1 mg ofa lipid sample to 2 ml of dimethyl carbonate:isooctane, 1:1. 1 ml of asolution containing 2.3 g sodium dissolved in 200 ml of methanol wasadded and the test tube was shaken vigorously for 30 s and left at roomtemperature for 15 min to ensure complete reaction. 3 ml water was addedand the test-tube was shaken and then centrifuged at 2×g. 0.5 μl of theorganic layer was injected on the chromatograph with the followingseparation conditions. The oven was temperature programmed, starting at130° C. (2 min), increased to 150° C. (30°/min) and 220° C. (3.2°C./min) with a 10 min hold. The injector temperature was 130° C. and thedetector temperature was 250° C. Initially the gas flow was 2.7 ml/min.The results are expressed as normalized weight percentages using theexternal standard method. No correction factors are used for the minorconstituents for which standards are not available or acceptably pure.

                  TABLE 2                                                         ______________________________________                                        Characterisation of fatty acid composition                                    Fatty acid                                                                    composi-                                                                      tion,                                                                         weight %                                                                             o-GL      o-h-GL  o-DGDG w-GL w-DGDG r-GL                              ______________________________________                                        C 14:0                                                                        C 16:0 20     21     21    16     15   13     12                              C 18:0 1      1      74    2      1    1                                      C 18:1 n-9                                                                           17     17           19     6    5      8                               C 18:1 n-7                                                                           1      1            1      1    1      1                               C 18:2 n-6                                                                           53     52           58     71   68     69                              C 18:3 n-3                                                                           2      2            3      3    3      5                               Minors 6      6       5    1      3    8      5                               <1% and                                                                       unidentifi-                                                                   ed                                                                            ______________________________________                                    

NMR Spectroscopy of Digalactosyldiacylglycerols

One-dimensional proton-decoupled natural abundance ¹³ C NMR spectra wererecorded on a Bruker AM-400 spectrometer (Bruker Analytische MesstechnikGmbH., Germany) at a ¹³ C frequency of 100.614 MHz. The pulse angle was36°, the pulse repetition time 1.0 s and resolution 1.526 Hz per datapoint. 3 Hz line broadening was applied during processing. The samples(10-40 mg) were diluted in a mixture of 730 μl DMSO-d₆ (Aldrich ChemicalComp., Inc., USA) and 20 μl D₂ O (Aldrich Chemical Comp., Inc., USA) andtransferred to an NMR tube (5 mm i.d.).

                  TABLE 3                                                         ______________________________________                                        .sup.13 C Chemical shifts (ppm) of digalactosyldiacylglycerols from           wheat and oats                                                                Signal      w-DGDG        o-DGDG                                              ______________________________________                                        Fatty acid moieties                                                           C(n)        13.8          13.7                                                C(n-1)      21.9          21.9                                                C(n-2)      30.8          30.8                                                C, methylene                                                                              28.3-28.9     28.4-29.0                                           C, allylic  26.5          26.5                                                C, doubly allylic                                                                         25.1          25.1                                                C, olefinic 127.6-129.6   127.6-129.5                                         C3          24.3          24.3                                                C2          33.3, 33.5    33.3, 33.5                                          C1          172.2, 172.5  172.1, 172.4                                        Glycerol moiety                                                               sn-1        62.3          62.4                                                sn-2        69.8          69.8                                                sn-3        66.6          66.6                                                Digalactosyl moiety                                                           C1 (inner)  103.6         103.6                                               C1' (outer) 99.4          99.4                                                others      60.4, 66.3, 67.7, 68.2,                                                                     60.4, 66.3, 67.7, 68.2,                                         68.6, 69.3, 70.1, 71.1,                                                                     68.6, 69.3, 70.1, 71.1,                                         72.8, 72.8    72.8, 72.9                                          ______________________________________                                    

EXAMPLES

In the examples below commercially available chemicals were used withoutfurther purification if not otherwise stated. Deionised,membrane-filtered water was used in all preparations. Soybean oil andmedium-chain triacylglycerol (MCT) oil, fish oils and oils with highcontents of γ-linolenic acid (GLA), obtained from evening primroseseeds, were used as model oils. However, the type of oily matter is notcrucial to obtain the specific benefits of the present invention.

Soybean oil, corn oil, and MCT oil were manufactured by Karlshamns AB,Sweden, and chromatographically purified. Evening primrose oils withdifferent contents of GLA, free GLA, and fish oils were manufactured byCallanish Ltd., Scotland, and used as received, except the fish oilswhich were chromatographically purified.

The antioxidants ascorbyl palmitate and E 442 (ammonium phosphatides)were obtained from Roche Products Ltd., UK, and Palsgaard AS, Denmark,respectively.

The emulsions were prepared by high-pressure homogenisation, usingdifferent equipment as stated in the examples. The particle (droplet)size distributions and the zeta potential of the resulting emulsionswere determined by dynamic light scattering (Zetasizer 4; MalvernInstruments Ltd., UK) at room temperature. The particle sizemeasurements were carried out at an angle of 90°, using a AZ104 cell andmultimodal analysis. Data are reported as Z averages. Zeta potentialswere measured with the same cell with the following instrumentalsettings: Cross beam mode, F(ka)=1.50 and cell voltage 134 V.

Example 1 Preparation of a 10% Fat Emulsion (MCT Oil)

An oil-in-water emulsion (batch size 200 g) was prepared containing thefollowing ingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier     0.5                                                            MCT oil        10.0                                                           Glycerol, 99%  2.3                                                            Water          ad 100.0                                                       ______________________________________                                    

The emulsifier, that is the galactolipid, was dispersed in the oil.Glycerol and water were mixed. The oil phase and the aqueous phase werepreheated to 70° C. and 85° C., respectively. The aqueous phase wasadded to the oil phase under high shear mixing at 18,000 rpm for 6 min.The preemulsion was then homogenised at 80 MPa and 50° C. for 6 cycles(Mini-Lab 8.30 H; APV Rannie AS, Denmark). The emulsion formed had anaverage droplet size of 243 nm.

Example 2 Preparation of a 20% Fat Emulsion (MCT Oil)

An oil-in-water emulsion (batch size 200 g) was prepared containing thefollowing ingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier     1.0                                                            MCT oil        20.1                                                           Glycerol, 99%  2.3                                                            Water          ad 100.0                                                       ______________________________________                                    

The emulsifier, that is the galactolipid, was dispersed in the oil.Glycerol and water were mixed. The oil phase was preheated to 90° C. andthe aqueous phase to 50° C. The oil phase was added to the aqueous phaseunder high shear mixing at 14,000 rpm for 4 min. The preemulsion wasthen homogenised at 80 MPa and 45° C. for 5 cycles (Mini-Lab 8.30 H; APVRannie AS, Denmark). The emulsion formed had an average droplet size of213 nm. This average size was not significantly altered by autoclaving(121° C., 20 min) and shaking (120 h, 150 cycles/min).

Example 3 Preparation of a 30% Fat Emulsion (MCT Oil)

An oil-in-water emulsion (batch size 200 g) was prepared using thefollowing ingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier     1.5                                                            MCT oil        30.1                                                           Glycerol, 99%  2.3                                                            Water          ad 100.0                                                       ______________________________________                                    

The emulsifier, that is the galactolipid, was dispersed in the oil.Glycerol and water were mixed. The oil phase was preheated to 67° C. andthe aqueous phase to 55° C. The oil phase was added to the aqueous phaseduring high shear mixing at 13,000 rpm for 6 min. The preemulsion wasthen homogenised at 80 MPa and 40° C. for six cycles (Mini-Lab 8.30 H;APV Ranhie AS, Denmark), which resulted in an average droplet size of200 nm.

One part of the resulting emulsion was heat sterilized in a standardbench autoclave at 121° C. for 20 min. After the heat treatment adroplet size of 209 nm was determined, indicating that the emulsiondroplets were not significantly affected during the process.

Another part of the emulsion was exposed to a shaking test at 150cycles/min for 5 days. No aggregation of the emulsion droplets andsubsequent creaming could be observed after the shaking test. Theaverage droplet size, 206 nm, indicated that the emulsion was verystable against shaking at high frequency for a long period of time. Alsothe heat sterilized emulsion was exposed to the same shaking testwithout any noticeable change in test performance.

An emulsion based on 1.2% egg phospholipids and 20% soybean oil did notwithstand the shaking test at the same frequency; creaming could beobserved on the top of the egg phospholipid emulsion after 1-2 hours.

Example 4 Preparation of a 39% Fat Emulsion (MCT Oil)

An oil-in-water emulsion (batch size 200 g) was prepared using thefollowing ingredients:

    ______________________________________                                               Ingredient    %                                                        ______________________________________                                               Emulsifier                                                                              2.0                                                                 MCT oil   39.4                                                                Water     ad 100.0                                                     ______________________________________                                    

The emulsifier, that is the galactolipid, was dispersed in the oil.Water and the oil phase were preheated to 70° C. and the oil was addedto the aqueous phase under high shear mixing at 16,000 rpm for 7 min.The preemulsion was then homogenised at 82 MPa and 50° C. for 6 cycles(Mini-Lab 8.30 H; APV Ranhie AS, Denmark). This formulation gave anemulsion with a slightly creamy consistency and a narrow sizedistribution with an average droplet size of 206 nm.

Example 5 Preparation of a 50% Fat Emulsion (MCT Oil)

An oil-in-water emulsion (batch size 200 g) was prepared containing thefollowing ingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier                   2.5                                              MCT oil                      50.3                                             Glycerol, 99%                2.3                                              Water                 ad    100.0                                             ______________________________________                                    

The emulsifier, that is the galactolipid, was dispersed in the oil.Glycerol and water were mixed. The oil phase was preheated to 60° C. andthe aqueous phase to 75° C. The oil phase was added to the aqueous phaseunder high shear mixing at 20,000 rpm for 4.5 min. The preemulsion wasthen homogenised at 80 MPa and 55° C. for 5 cycles (Mini-Lab 8.30 H; APrRannie AS, Denmark). The emulsion formed was quite high in viscosity("yoghurt-like") with an average droplet size of 235 nm.

Example 6 Preparation of a 20% Fat Emulsion (MCT/Soybean Oil)

An oil-in-water emulsion (batch size 200 g) was prepared containing thefollowing ingredients:

    ______________________________________                                        Ingredient              %                                                     ______________________________________                                        Galactolipid material          1.0                                            Phosphatidylcholine from soybean                                                                             1.0                                            Soybean oil                    10.0                                           MCT oil                        10.0                                           Glycerol, 99%                  2.3                                            Water                   ad    100.0                                           ______________________________________                                    

The galactolipid material and soybean phosphatidylcholine were dispersedin the oil mixture. Glycerol and water were mixed. The oil phase waspreheated to 65° C. and the aqueous phase to 55° C. The aqueous phasewas added to the oil phase under high shear mixing at 11,000 rpm for 9min. The preemulsion was then homogenised at 80 MPa and 46° C. for 5cycles (Mini-Lab 8.30 H; APV Rannie AS, Denmark). The emulsion formedhad an average droplet size of 262 nm which did not change significantlyafter autoclaving.

Example 7 Preparation of a 20% Fat Emulsion (Soybean Oil)

An oil-in-water emulsion (batch size 200 g) was prepared using thefollowing ingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier                   1.5                                              MCT oil                      20.0                                             Glycerol, 99%                2.3                                              Water                 ad    100.0                                             ______________________________________                                    

The emulsifier, that is the galactolipid, was dispersed and hydrated ina portion of the water. Glycerol and the rest of the water was thenadded and mixed. The aqueous dispersion was subjected to high pressurehomogenisation for 2 cycles at 60 MPa and 40° C. The soybean oil,preheated to 40° C., was added to the aqueous dispersion under highshear mixing at 13,000 rpm for 10 min. The preemulsion was thenhomogenised at 80 MPa and 40° C. for 6 cycles (Mini-Lab 8.30 H; APVRannie AS, Denmark). After cooling to room temperature, the emulsion wasadjusted to pH 7.2 using a 2M NaOH solution.

Table 4 summarizes the average droplet size in nm of the emulsionsdescribed in Examples 1-7.

In addition zeta potential values in mV are listed in Table 4 indicatingthat the emulsion droplets carried a significant negative charge whichimplies a good shelf life of the emulsions.

                  TABLE 4                                                         ______________________________________                                                                  After   After                                                                              Zeta                                   Ex.  Oily        Initial  autocla-                                                                              shake                                                                              poten-                                 no.  material    emulsion ving    test tial                                   ______________________________________                                        1    10% MCT oil 243                   -69                                    2    20% MCT oil 213      226     222  -72                                    3    30% MCT oil 200      209     206  -68                                    4    39% MCT oil 206      216          -71                                    5    50% MCT oil 235                   -72                                    6    10% MCT oil 262      266          -69                                         10% soybean                                                                   oil                                                                      7    20% soybean 400                   -77                                         oil                                                                      ______________________________________                                    

Example 8 Preparation of a 20% Fat Emulsion (Evening Primrose OilContaining 9% GLA)

An oil-in-water emulsion (batch size 200 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier, hydrogenated     1.02                                             Evening primrose oil         20.46                                            Water                 ad    100.00                                            ______________________________________                                    

The emulsifier, that is the hydrogenated galactolipid, was dispersed inthe oil. The oil phase and water were preheated to 62° C. and 73 ° C.,respectively, and the oil phase was added to the water under high shearmixing at 14,000 rpm for 2.5 min. The preemulsion was then homogenisedat 80 MPa and 56° C. for 7 cycles (Mini-Lab 8.30 H; APr Rannie AS,Denmark). This formulation gave an emulsion with an average droplet sizeof 240 nm. The zeta potential was -57 mV.

Example 9 Preparation of a 20% Fat Emulsion (Evening Primrose OilContaining 9% GLA)

An oil-in-water emulsion (batch size 200 g) was prepared with followingingredients:

    ______________________________________                                        Ingredient            %                                                       ______________________________________                                        Emulsifier, enriched         1.01                                             Evening primrose oil         20.16                                            Water                 ad    100.00                                            ______________________________________                                    

The emulsifier, that is the enriched galactolipid, was dispersed in theoil. The oil phase and water were preheated to 64° C. and 63° C.,respectively, and the oil phase was added to the water under high-shearmixing at 13,500 rpm for 2.5 min. The preemulsion was then homogenisedat 80 MPa and 50° C. for 7 cycles (Mini-Lab 8.30 H; APV Ranhie AS,Denmark). This formulation gave an emulsion with an average droplet sizeof 260 nm and a zeta potential of -50 mV.

Example 10 Preparation of a 40% Fat Emulsion (Evening Primrose OilContaining 9% GLA)

An oil-in-water emulsion (batch size 300 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient              %                                                     ______________________________________                                        Emulsifier                     1.99                                           Evening primrose oil           39.55                                          Vitamin E acetate              1.08                                           Ammonium phosphatides, E 442   0.10                                           Ascorbyl palmitate             0.02                                           Sucrose                        14.08                                          Lemon flavour                  2.00                                           Potassium sorbate              0.10                                           Citric acid                    0.01                                           Water                   ad    100.0                                           ______________________________________                                    

The emulsifier and antioxidants were dispersed in the oil. Sucrose,preservative, flavour and water were mixed. The oil phase and theaqueous phase were preheated to 60° C. and 68° C., respectively, and theoil was added to the aqueous phase under high shear mixing at 17,000 rpmfor 4 min. The preemulsion was then homogenised at 80 MPa and 60° C. for5 cycles (Mini-Lab 8.30 H; APV Rannie AS, Denmark). This formulationgave an emulsion with an average droplet size of 230 nm and a zetapotential of -72 mV. The pH was 5.8.

Example 11 Preparation of a 36% Fat Emulsion (Evening Primrose OilContaining 9% GLA)

An oil-in-water emulsion (batch size 2300 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient              %                                                     ______________________________________                                        Emulsifier                     1.80                                           Evening primrose oil           35.97                                          Vitamin E acetate              1.09                                           Ammonium phosphatides, E 442   0.10                                           Ascorbyl palmitate             0.02                                           Sucrose                        15.00                                          Banana flavour                 2.00                                           Potassium sorbate              0.10                                           Water                   ad    100.0                                           ______________________________________                                    

The emulsifier and antioxidants were dispersed in the oil. Sucrose,preservative, flavour and water were mixed. The oil phase and theaqueous phase were preheated to 58° C. and 63° C., respectively, and theoil was added to the aqueous phase under high shear mixing at 16,000 rpmfor 7.5 min. The preemulsion was then homogenised (Model LAB, Type 12.51H; APV Rannie AS, Denmark) at a total pressure of 50 MPa and a pressureof 10 MPa over the second stage. The flow was 0.82 l/min, the total time12 min and the temperature 48° C. This formulation gave an emulsion withan average droplet size of 230 nm and a zeta potential of -72 mV.

Example 12 Preparation of a 40% Fat Emulsion (Enriched Evening PrimroseOil Containing 20% GLA)

An oil-in-water emulsion (batch size 300 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient               %                                                    ______________________________________                                        Emulsifier                      2.49                                          Enriched evening primrose oil, 20% GLA                                                                        39.85                                         Vitamin E acetate               0.39                                          Ammonium phosphatides, E 442    0.10                                          Ascorbyl palmitate              0.02                                          Sucrose                         15.04                                         Lemon flavour                   2.00                                          Potassium sorbate               0.10                                          Water                    ad    100.0                                          ______________________________________                                    

The emulsifier and antioxidants were dispersed in the oil. Sucrose,preservative, flavour and water were mixed. Both phases were preheatedto 65°-70° C. and the oil was added to the aqueous phase under highshear mixing at 15,000 rpm for 3.5 min. The preemulsion was thenhomogenised at 80 MPa and 60° C. for 5 cycles (Mini-Lab 8.30 H; APrRannie AS, Denmark). This formulation gave an emulsion with a thickyoghurt-like consistency.

Example 13 Preparation of an 11% Fat Emulsion (Enriched Evening PrimroseOil Containing 80% GLA)

100 g of an oil-in-Water emulsion was prepared-containing the followingingredients:

    ______________________________________                                        Ingredient             %                                                      ______________________________________                                        Emulsifier, enriched   1.0                                                    Enriched evening                                                              primrose oil, 80% GLA  11.0                                                   Glycerol, 2.3% in water                                                                              ad 100.0                                               ______________________________________                                    

The emulsifier, that is the enriched galactolipid material, wasdissolved in the oil at approximately 50° C. under nitrogen. Glyceroland water were mixed. The aqueous phase was added to the oil phase underhigh shear mixing at 12,000 rpm for 30 s. The preemulsion was heated to35° C. and homogenised at 83 MPa for 5 min (EmulsiFlex-C30, AvestinInc., Canada). The resulting emulsion had an average droplet size of 224nm, a zeta potential of -40 mV, and was easily filtered through amembrane filter with a pore size of 0.22 μm.

Example 14 Preparation of a 20% Fat Emulsion (Free Fatty Acid Containing70% GLA)

50 g of an oil-in-water emulsion was prepared containing the followingingredients:

    ______________________________________                                        Ingredient             %                                                      ______________________________________                                        Emulsifier, enriched   2.5                                                    Free fatty acid, 70% GLA                                                                             20.0                                                   Glycerol, 2.3% in water                                                                              ad 100.0                                               ______________________________________                                    

The emulsifier, that is the enriched galactolipid material, wasdissolved in the free fatty acid at approximately 50° C. under nitrogen.Glycerol and water were mixed. The aqueous phase was added to the oilphase under high shear mixing at 12,000 rpm for 30 s. The preemulsionwas heated to 35° C. and homogenised at 86 MPa for 6.5 min(EmulsiFlex-C30, Avestin Inc., Canada). The resulting emulsion had anaverage droplet size of 211 nm, a zeta potential of -40 mV, and waseasily filtered through a membrane filter with a pore size of 0.22 μm.

Example 15 Preparation of a 39% Fat Emulsion (Sardine Oil inEicosapentaenoic Acid (EPA))

An oil-in-water emulsion (batch size 250 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient             %                                                      ______________________________________                                        Emulsifier             3.88                                                   Sardine oil            38.93                                                  Vitamin E acetate      1.08                                                   Ammonium phosphatides, E 442                                                                         1.00                                                   Ascorbyl palmitate     0.02                                                   Sucrose                14.98                                                  Peppermint flavour     1.00                                                   Potassium sorbate      0.20                                                   Water                  ad 100.0                                               ______________________________________                                    

The emulsifier and antioxidants were dispersed in the oil. Sucrose,preservative, flavour and water were mixed. The oil phase and theaqueous phase were preheated to 57° C. and 51° C. respectively and theoil was added to the aqueous phase under high shear mixing at 16,000 rpmfor 3.5 min. The preemulsion was then homogenised at 80 MPa and 55° C.for 7 cycles (Mini-Lab 8.30 H; APV Rannie AS, Denmark). This formulationgave an emulsion with an average droplet size of 190 nm and a zetapotential of -72 mV.

Example 16 Preparation of a 39% Fat Emulsion (Tuna Fish Oil Rich inDocosahexanoic Acid (DHA)

An oil-in-water emulsion (batch size 250 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient             %                                                      ______________________________________                                        Emulsifier             3.91                                                   Tuna fish oil          39.08                                                  Vitamin E acetate      1.10                                                   Ammonium phosphatides, E 442                                                                         1.00                                                   Ascorbyl palmitate     0.02                                                   Sucrose                14.94                                                  Peppermint flavour     1.00                                                   Potassium sorbate      0.20                                                   Water                  ad 100.0                                               ______________________________________                                    

The emulsifier and antioxidants were dispersed in the oil. Sucrose,preservative, flavour and water were mixed. The oil phase and theaqueous phase were preheated to 59° C. and 64° C. respectively and theoil was added to the aqueous phase under high shear mixing at 16,000 rpmfor 5 min. The preemulsion was then homogenised at 80 MPa and 60° C. for7 cycles (Mini-Lab 8.30 H; APV Rannie AS, Denmark). This formulationgave an emulsion with an average droplet size of 190 nm and a zetapotential of -75 mV.

Example 17 Preparation of a 40% Fat Emulsion (Corn Oil)

An oil-in-water emulsion (batch size 200 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredient             %                                                      ______________________________________                                        Emulsifier             2.00                                                   Corn oil               40.08                                                  Ammonium phosphatides, E 442                                                                         1.00                                                   Ascorbyl palmitate     0.02                                                   Sucrose                14.98                                                  Potassium sorbate      0.10                                                   Water                  ad 100.0                                               ______________________________________                                    

The emulsifier and antioxidants were dispersed in the oil. Sucrose,preservative, and water were mixed. Both phases were preheated to 65° C.and the oil was added to the aqueous phase under high shear mixing at15,000 rpm for 4 min. The preemulsion was then homogenised at 80 MPa and55° C. for 7 cycles (Mini-Lab 8.30 H; APV Rannie AS, Denmark). Thisformulation gave an emulsion with a narrow size distribution and anaverage droplet size of 210 nm and a zeta potential of -74 mV.

Example 18 Preparation of a 11% Parenteral Fat Emulsion (Soybean Oil)Containing 2,6-Diisopropylphenol

An oil-in-water emulsion (batch size 150 g) containing apharmacologically active compound was prepared using the followingingredients:

    ______________________________________                                        Ingredient             %                                                      ______________________________________                                        Emulsifier             1.27                                                   Soybean oil            10.57                                                  2,6-diisopropylphenol  1.05                                                   Glycerol, 99%          2.24                                                   Water                  ad 100.00                                              ______________________________________                                    

The emulsifier, that is the galactolipid, and the active ingredient, ananaesthetic drug, were dissolved in the soybean oil. Glycerol and waterwere mixed. The aqueous dispersion and the drug-containing oil phasewere preheated to 72° C. and 68° C., respectively. The oil phase wasadded to the aqueous dispersion under high shear mixing at 13,000 rpmfor 1.5 min. The preemulsion was then homogenised at 80 MPa and 48° C.for 7 cycles (Mini-Lab 8.30 H; APV Ranhie AS, Denmark). The emulsionformed had an average droplet size of 170 nm and a zeta potential of -63mV. The osmolality determined with a microosmometer (Type 13; HermannRoebling Messtechnik, Germany) was 257 milliosmol/kg H₂ O.

Conclusions

Our findings related to the invention are that it is possible to produceremarkably stable oil-in-water emulsions based on the galactolipidmaterial, which fulfils the important and necessary requirements ofbeing autoclavable and resistant to harsh mechanical treatments. Theemulsions have particle size distributions which are suitable forparenteral and intravenous use. The emulsions do not exhibit anyunpleasant odour or taste and they are remarkably stable towardsoxidation. This invention provides an alternative to the phospholipidemulsions which offers concrete advantages compared to such emulsions.

We claim:
 1. An oil-in water emulsion comprising an emulsifier in anamount of about 0.01-50% by weight of the total preparation, an oilymaterial emulsified in a polar solvent in an amount of about 0.1-70% byweight of the total preparation, and polar lipids in the remainingamount of the total preparation,wherein the emulsifier is a galactolipidmaterial consisting of at least 50% digalactosyldiacylglycerols, andwherein the ratio of the emulsifier and the oily material is about 1:10to about 1:40 by weight.
 2. An emulsion according to claim 1, whereinthe galactolipid material consists of about 70-80%digalactosyldiacylglycerols and about 20-30% other polar lipids.
 3. Anemulsion according to claim 1, wherein the galactolipid materialconsists up to 100% digalactosyldiacylglycerols.
 4. An emulsionaccording to claim 1, wherein the oily material comprises γ-linolenicacid in the form of a free acid, its salts or esters.
 5. An emulsionaccording to claim 1, wherein the oily material is an evening primroseoil or a borago oil.
 6. A pharmaceutical, nutritional or cosmeticcomposition which comprises the emulsion of claim 1 as a carrier for anactive substance.
 7. A pharmaceutical composition comprising anoil-in-water emulsion according to claim 1 further comprises atherapeutically active substance.
 8. A pharmaceutical compositionaccording to claim 7, wherein the oily material comprises γ-linolenicacid in the form of a free acid, its salts or esters.
 9. Apharmaceutical composition according to claim 7, wherein the oilymaterial is an evening primrose oil or a borago oil.
 10. Apharmaceutical composition according to claim 7, wherein the oilymaterial is a triacylglycerol oil.
 11. A pharmaceutical compositionaccording to claim 7, comprising:a therapeutically active substance in atherapeutically effective amount; a galactolipid emulsifier in an amountof about 0.1-5.0% by weight of the total composition; an oily materialin an amount of about 1-50% by weight of the total composition;optionally, an isotonic agent in an isotonically effective amount; and apolar solvent.
 12. A pharmaceutical composition for parenteraladministration according to claim 10, consisting of, by weight of thetotal composition,about 0.2-3% 2,6-diisopropylphenol, about 0.3-5%galactolipid material, about 5-30% triacylglycerol oil, about anisotonically effective amount of an isotonic agent, and about 100%water.
 13. A pharmaceutical composition according to claim 7 for oral,enteral, parenteral, rectal, vaginal, topical, ocular, nasal or auraladministration.
 14. An oil-in-water emulsion according to claim 1wherein the emulsifier is in an amount of about 0.1-10% by weight of thetotal preparation.
 15. A pharmaceutical composition according to claim7, wherein the oily material is a medium-chain triacylglycerol oil or abioactive substance.
 16. An oil-in-water emulsion according to claim 1wherein the ratio of the emulsifier and the oily material is about 1:15to about 1:25 by weight.